The creation of a powerful new non-linear laser microscopy for biomedical research and pathology investigations is the objective of this research. New and convenient instrumentation for imaging the dynamic microchemistry of individual living cells and tissues is to be developed from the recent invention: "Two Photon Excitation Laser Scanning Fluorescence Microscopy" (Denk, Strickler and Webb, Science 248:73-76,1990). Specific projects include measurement of two photon excitation spectra of fluorophores, engineering design for the non-linear optical system including adaptation of economical solid state femtosecond lasers and integration of powerful analytic control computers, development of new methods of measurement of three-dimensional molecular diffusion (as in the cellular cytoplasm) enabled by the inherent three-dimensional resolution of two-photon excitation and photolysis, measure molecular mechanisms of photolysis, photobleaching and cellular photodamage with the help of high intensity laser pulses, and develop imaging of fluorescence decay time data for cellular microenvironmental analysis. Introduction of non-linear physical optics into optical microscopy offers an opportunity for major advance in analytical capability for quantitative biophysical instrumentation that this research aims to realize.